Synthesis and application of fluorescent probe for detecting hypochlorous acid

Abstract

The invention relates to the technical field of biochemical materials, discloses synthesis and application of a fluorescent probe for detecting hypochlorous acid and a detection method of hypochlorite, and relates to the technical field of biochemical materials. The hypochlorite fluorescent probe provided by the invention is high in sensitivity to hypochlorite ions, good in selectivity, high in response speed and accurate in detection result. Compared with an existing hypochlorous acid fluorescent probe, the hypochlorite fluorescent probe provided by the invention is simple in preparation method, cheap in detection reagent, simple, convenient and rapid in detection process and suitable for popularization and application.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical materials, in particular to the synthesis and application of a fluorescent probe for detecting hypochlorous acid. Background Art

[0002] A fluorescent probe for detecting hypochlorous acid is a novel hypochlorous acid fluorescent probe PIOCl conjugated with phenothiazine-indanone. In biological cells, hypochlorous acid is usually produced by the reaction of hydrogen peroxide and chloride ions under the catalysis of myeloperoxidase (MPO). It can react with a variety of biological molecules such as proteins, DNA, RNA, fatty acids and cholesterol, participate in many physiological and pathological processes, and play a vital role in maintaining the redox balance in cells. Hypochlorous acid is of great significance in innate host defense and inflammatory response. It exerts antimicrobial effects under physiological conditions and plays a role in protecting the body. Studies have shown that hypochlorous acid is also a natural adaptive immune adjuvant. However, under certain conditions, it has been confirmed that oxidative stress caused by excessive hypochlorous acid is related to a series of diseases, such as arthritis, leukemia, nephritis, small vessel inflammation, tumors and atherosclerosis. Therefore, it is a major issue and of great significance to be able to detect the changes in hypochlorous acid concentration in organisms and the environment in a timely and effective manner. At present, the detection technologies of hypochlorous acid include fluorescence, electrochemical, colorimetric, bio- and chemiluminescent methods. Fluorescence detection has attracted more and more attention due to its advantages such as high sensitivity, good selectivity and in-situ detection. With the deepening of research, more hypochlorous acid fluorescent probes based on different fluorescent groups may be developed in the future to meet different detection needs and environments. These new probes will further promote the development of biomedical research and clinical diagnostic technology. In general, as an efficient hypochlorous acid fluorescent probe, the new probe is not only of great value in scientific research, but also has broad application prospects in clinical diagnosis and biological imaging. Summary of the invention

[0003] 1. Technical issues to be resolved

[0004] In view of the deficiencies in the prior art, the present invention provides a synthesis and application of a fluorescent probe for detecting hypochlorous acid, which has the characteristics of fast response and high sensitivity.

[0005] (II) Technical solution

[0006] To achieve the above object, the present invention provides the following technical solutions: a synthesis of a fluorescent probe for detecting hypochlorous acid and its application, a hypochlorite fluorescent probe having a structure shown in Formula I:

[0007]

[0008] Preferably, the preparation method of the hypochlorite fluorescent probe comprises the following steps:

[0009] (1) mixing 4-pyridineacetonitrile and 2-bromoethanol to carry out a nucleophilic substitution reaction to obtain a compound having a structure shown in Formula II;

[0010]

[0011] (2) mixing the compound having the structure shown in Formula II with 9-anthracene carboxaldehyde and a base catalyst, and performing a Knoevenagel condensation reaction to obtain a hypochlorite fluorescent probe having the structure shown in Formula I;

[0012]

[0013] Preferably, the molar ratio of 4-pyridineacetonitrile to 2-bromoethanol in (1) is 1:1-3.

[0014] Preferably, the temperature of the nucleophilic substitution reaction in (1) is 70-75° C., and the time of the nucleophilic substitution reaction is 6-8 h.

[0015] Preferably, the molar ratio of the compound having the structure shown in Formula II to 9-anthracenecarboxaldehyde in (2) is 1:1-2.

[0016] Preferably, the temperature of the Knoevenagel condensation reaction is below 70° C., and the time of the Knoevenagel condensation reaction is 5-6 h.

[0017] Preferably, the method comprises the following steps: mixing the hypochlorite fluorescent probe according to claim 1 or the hypochlorite fluorescent probe prepared by the preparation method according to any one of claims 2 to 6 with a sample to be tested, and performing fluorescence detection.

[0018] Preferably, the method of mixing the hypochlorite fluorescent probe and the sample to be tested comprises:

[0019] S1: dissolving the hypochlorite fluorescent probe in dimethyl sulfoxide to obtain a fluorescent probe stock solution;

[0020] S2: mixing the fluorescent probe stock solution and the acetonitrile / PBS buffer solution system to obtain a fluorescent probe detection solution; and adding a sample to be tested into the fluorescent probe detection solution.

[0021] Preferably, the fluorescence detection includes fluorescence qualitative detection and fluorescence quantitative detection.

[0022] (III) Beneficial technical effects

[0023] The present invention relates to the technical field of biochemical materials by synthesizing a fluorescent probe for detecting hypochlorous acid and its application, and a method for detecting hypochlorite. The hypochlorite fluorescent probe provided by the present invention has high sensitivity to hypochlorite ions, good selectivity, fast response speed, and accurate detection results. Compared with the existing hypochlorite fluorescent probe, the preparation method of the hypochlorite fluorescent probe provided by the present invention is simple, the detection reagent is cheap, the detection process is simple and fast, and it is suitable for popularization and application. DETAILED DESCRIPTION

[0024] Example 1

[0025] (1) Mixing 4-pyridineacetonitrile and 2-bromoethanol, wherein the molar ratio of 4-pyridineacetonitrile to 2-bromoethanol is 1:1, and performing a nucleophilic substitution reaction at a temperature of 70° C. and a time of 6 hours to obtain a compound having a structure shown in Formula II;

[0026] (2) Mixing the compound having the structure shown in Formula II with 9-anthracene carboxaldehyde and a base catalyst, wherein the molar ratio of the compound having the structure shown in Formula II to 9-anthracene carboxaldehyde is 1:1, and performing a Knoevenagel condensation reaction, wherein the temperature of the Knoevenagel condensation reaction is below 70° C., and the Knoevenagel condensation reaction time is 5 hours. Obtaining a hypochlorite fluorescent probe having the structure shown in Formula I;

[0027] (3) mixing the prepared hypochlorite fluorescent probe with a sample to be tested and performing fluorescence detection, wherein the fluorescence detection includes fluorescence qualitative detection and fluorescence quantitative detection;

[0028] (4) dissolving the hypochlorite fluorescent probe in dimethyl sulfoxide to obtain a fluorescent probe stock solution;

[0029] (5) Mixing the fluorescent probe stock solution and the acetonitrile / PBS buffer solution system to obtain a fluorescent probe detection solution; and adding the sample to be tested into the fluorescent probe detection solution.

[0030] Example 2

[0031] (1) Mixing 4-pyridineacetonitrile and 2-bromoethanol, wherein the molar ratio of 4-pyridineacetonitrile to 2-bromoethanol is 1:3, and performing a nucleophilic substitution reaction at a temperature of 75° C. for 8 hours to obtain a compound having a structure shown in Formula II;

[0032] (2) Mixing the compound having the structure shown in Formula II with 9-anthracene carboxaldehyde and a base catalyst, wherein the molar ratio of the compound having the structure shown in Formula II to 9-anthracene carboxaldehyde is 1:2, and performing a Knoevenagel condensation reaction, wherein the temperature of the Knoevenagel condensation reaction is below 70° C., and the Knoevenagel condensation reaction time is 6 hours. Obtaining a hypochlorite fluorescent probe having the structure shown in Formula I;

[0033] (3) mixing the prepared hypochlorite fluorescent probe with a sample to be tested and performing fluorescence detection, wherein the fluorescence detection includes fluorescence qualitative detection and fluorescence quantitative detection;

[0034] (4) dissolving the hypochlorite fluorescent probe in dimethyl sulfoxide to obtain a fluorescent probe stock solution;

[0035] (5) Mixing the fluorescent probe stock solution and the acetonitrile / PBS buffer solution system to obtain a fluorescent probe detection solution; and adding the sample to be tested into the fluorescent probe detection solution.

[0036] Example 3

[0037] (1) Mixing 4-pyridineacetonitrile and 2-bromoethanol, wherein the molar ratio of 4-pyridineacetonitrile to 2-bromoethanol is 1:2, and performing a nucleophilic substitution reaction at a temperature of 72° C. and a time of 7 hours to obtain a compound having a structure shown in Formula II;

[0038] (2) Mixing the compound having the structure shown in Formula II with 9-anthracene carboxaldehyde and a base catalyst, wherein the molar ratio of the compound having the structure shown in Formula II to 9-anthracene carboxaldehyde is 1:1.5, and performing a Knoevenagel condensation reaction, wherein the temperature of the Knoevenagel condensation reaction is below 70° C., and the Knoevenagel condensation reaction time is 5.5 h. Obtaining a hypochlorite fluorescent probe having the structure shown in Formula I;

[0039] (3) mixing the prepared hypochlorite fluorescent probe with a sample to be tested and performing fluorescence detection, wherein the fluorescence detection includes fluorescence qualitative detection and fluorescence quantitative detection;

[0040] (4) dissolving the hypochlorite fluorescent probe in dimethyl sulfoxide to obtain a fluorescent probe stock solution;

[0041] (5) Mixing the fluorescent probe stock solution and the acetonitrile / PBS buffer solution system to obtain a fluorescent probe detection solution; and adding the sample to be tested into the fluorescent probe detection solution.

[0042] Example 4

[0043] (1) Mixing 4-pyridineacetonitrile and 2-bromoethanol, wherein the molar ratio of 4-pyridineacetonitrile to 2-bromoethanol is 1:1, and performing a nucleophilic substitution reaction at a temperature of 70° C. and a time of 6 hours to obtain a compound having a structure shown in Formula II;

[0044] (2) Mixing the compound having the structure shown in Formula II with 9-anthracene carboxaldehyde and a base catalyst, wherein the molar ratio of the compound having the structure shown in Formula II to 9-anthracene carboxaldehyde is 1:1, and performing a Knoevenagel condensation reaction, wherein the temperature of the Knoevenagel condensation reaction is below 70° C., and the Knoevenagel condensation reaction time is 5 hours. Obtaining a hypochlorite fluorescent probe having the structure shown in Formula I;

[0045] (3) mixing the prepared hypochlorite fluorescent probe with a sample to be tested and performing fluorescence detection, wherein the fluorescence detection includes fluorescence qualitative detection and fluorescence quantitative detection;

[0046] (4) dissolving the hypochlorite fluorescent probe in dimethyl sulfoxide to obtain a fluorescent probe stock solution;

[0047] (5) Mixing the fluorescent probe stock solution and the acetonitrile / PBS buffer solution system to obtain a fluorescent probe detection solution; and adding the sample to be tested into the fluorescent probe detection solution.

[0048] Example 5

[0049] (1) Mixing 4-pyridineacetonitrile and 2-bromoethanol, wherein the molar ratio of 4-pyridineacetonitrile to 2-bromoethanol is 1:3, and performing a nucleophilic substitution reaction at a temperature of 75° C. for 8 hours to obtain a compound having a structure shown in Formula II;

[0050] (2) Mixing the compound having the structure shown in Formula II with 9-anthracene carboxaldehyde and a base catalyst, wherein the molar ratio of the compound having the structure shown in Formula II to 9-anthracene carboxaldehyde is 1:2, and performing a Knoevenagel condensation reaction, wherein the temperature of the Knoevenagel condensation reaction is below 70° C., and the Knoevenagel condensation reaction time is 6 hours. Obtaining a hypochlorite fluorescent probe having the structure shown in Formula I;

[0051] (3) mixing the prepared hypochlorite fluorescent probe with a sample to be tested and performing fluorescence detection, wherein the fluorescence detection includes fluorescence qualitative detection and fluorescence quantitative detection;

[0052] (4) dissolving the hypochlorite fluorescent probe in dimethyl sulfoxide to obtain a fluorescent probe stock solution;

[0053] (5) Mixing the fluorescent probe stock solution and the acetonitrile / PBS buffer solution system to obtain a fluorescent probe detection solution; and adding the sample to be tested into the fluorescent probe detection solution.

[0054] Comparative Example 1

[0055] The difference between this comparative example and Example 5 is that no fluorescent probe of hypochlorous acid is added.

[0056] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A hypochlorite fluorescent probe, characterized in that: The structure has the formula I:

2. The method for preparing the hypochlorite fluorescent probe according to claim 1, characterized in that: The preparation method of the hypochlorite fluorescent probe comprises the following steps: (1) mixing 4-pyridineacetonitrile and 2-bromoethanol to carry out a nucleophilic substitution reaction to obtain a compound having a structure shown in Formula II; (2) mixing the compound having the structure shown in Formula I with 9-anthracene carboxaldehyde and a base catalyst, and performing a Knoevenagel condensation reaction to obtain a hypochlorite fluorescent probe having the structure shown in Formula I; 3. The method for preparing the fluorescent probe for detecting hypochlorous acid according to claim 2, characterized in that: The molar ratio of 4-pyridineacetonitrile to 2-bromoethanol in (1) is 1:1-3.

4. The method for preparing the fluorescent probe for detecting hypochlorous acid according to claim 2, characterized in that: The temperature of the nucleophilic substitution reaction in (1) is 70-75° C., and the time of the nucleophilic substitution reaction is 6-8 h.

5. The method for preparing the fluorescent probe for detecting hypochlorous acid according to claim 2, characterized in that: The molar ratio of the compound having the structure shown in Formula II to 9-anthracenecarboxaldehyde in (2) is 1:1-2.

6. The method for preparing the fluorescent probe for detecting hypochlorous acid according to claim 2, characterized in that: The temperature of the Knoevenagel condensation reaction is below 70° C., and the time of the Knoevenagel condensation reaction is 5-6 hours.

7. A method for detecting hypochlorite not for the purpose of diagnosing and / or treating a disease, characterized in that: The method comprises the following steps: mixing the hypochlorite fluorescent probe according to claim 1 or the hypochlorite fluorescent probe prepared by the preparation method according to any one of claims 2 to 6 with a sample to be tested, and performing fluorescence detection.

8. The synthesis and application of the fluorescent probe for detecting hypochlorous acid according to claim 7, characterized in that: The method for mixing the hypochlorite fluorescent probe and the sample to be tested comprises: S1: dissolving the hypochlorite fluorescent probe in dimethyl sulfoxide to obtain a fluorescent probe stock solution; S2: mixing the fluorescent probe stock solution and the acetonitrile / PBS buffer solution system to obtain a fluorescent probe detection solution; and adding a sample to be tested into the fluorescent probe detection solution.

9. The synthesis and application of the fluorescent probe for detecting hypochlorous acid according to claim 7, characterized in that: The fluorescence detection includes fluorescence qualitative detection and fluorescence quantitative detection.

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